B‐cell lymphoma‐2 family proteins‐activated proteases as potential therapeutic targets for influenza A virus and severe acute respiratory syndrome coronavirus‐2: Killing two birds with one stone?

Abstract The COVID‐19 pandemic caused by severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2) has led to a global health emergency. There are many similarities between SARS‐CoV‐2 and influenza A virus (IAV); both are single‐stranded RNA viruses infecting airway epithelial cells and have similar modes of replication and transmission. Like IAVs, SARS‐CoV‐2 infections poses serious challenges due to the lack of effective therapeutic interventions, frequent appearances of new strains of the virus, and development of drug resistance. New approaches to control these infectious agents may stem from cellular factors or pathways that directly or indirectly interact with viral proteins to enhance or inhibit virus replication. One of the emerging concepts is that host cellular factors and pathways are required for maintaining viral genome integrity, which is essential for viral replication. Although IAVs have been studied for several years and many cellular proteins involved in their replication and pathogenesis have been identified, very little is known about how SARS‐CoV‐2 hijacks host cellular proteins to promote their replication. IAV induces apoptotic cell death, mediated by the B‐cell lymphoma‐2 (Bcl‐2) family proteins in infected epithelia, and the pro‐apoptotic members of this family promotes viral replication by activating host cell proteases. This review compares the life cycle and mode of replication of IAV and SARS‐CoV‐2 and examines the potential roles of host cellular proteins, belonging to the Bcl‐2 family, in SARS‐CoV‐2 replication to provide future research directions.


| INTRODUCTION
The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) emerged as the infectious agent responsible for the coronavirus disease 2019 (COVID-19) pandemic, and much like the influenza A virus (IAV), it primarily targets the human respiratory system. 1 Diseases associated with both IAV and SARS-CoV-2 infections vary from mild respiratory illness, to acute pneumonia, and even respiratory failure. 2 Historically, four IAV pandemics have been registered, amongst which the 1918 H1N1 'Spanish flu' was the most devastating, claiming approximately 50 million lives globally. 3 The recent flu pandemic caused by the H1N1/pdm09 virus, was reported in 2009 in 207 countries and resulted in 42-86 million infections. 4 This novel IAV has continued to spread as a seasonal epidemic which significantly impacts global health with an annual burden of around 3-5 million cases of severe illness and about 290,000-650,000 mortalities globally as per the world health organization (WHO) reports. 5 Additionally, a highly pathogenic avian H5N1 IAV has spread throughout Africa, Asia, and Europe by crossing over host species barriers to infect humans and cause mortalities. 6,7,8,9 Further to the emergencies of SARS caused by a Betacoronavirus were first reported in Wuhan, China in December 2019. 12 The patients were characterised by acute pneumonia-associated symptoms, such as chills, dry cough, fever, muscle pain, and shortness of breath. 13 The SARS-CoV-2 rapidly spread globally and as of 11 November 2022, there have been 630,832,131 confirmed cases of COVID-19, including 6,584,104 deaths, reported to the WHO. 14 In comparison with the other two coronaviruses, SARS-CoV-2 is much more contagious and infectious, which rapidly resulted in a pandemic that constituted a global health emergency.
As of 9 November 2022, a total of 12,885,748,541 anti-SARS-CoV-2 vaccine doses have been administered globally. 14 These vaccines have been responsible to prevent infection as well as adverse effects caused by several SARS-CoV-2 variants, possibly by reducing both asymptomatic and symptomatic infections, thereby preventing the onward transmission and reducing the viral loads. 15 However, similar viral loads found recently in unvaccinated and vaccinated people infected with various SARS-CoV-2 variants has raised questions about the effectiveness of the vaccines to prevent transmission. 16 Although the vaccines have managed to provide continued protection against infection, the degree of effectiveness varies among the variants, since newer variants, such as delta and omicron tend to erode vaccine-mediated protection. 17,18 This challenge necessitated research that focusses on the development of variant-adapted booster vaccines that can induce higher and broader immune responses against SARS-CoV-2. 19,20,21 Hence, identifying the host cellular targets for treating infections caused by both IAV and SARS-CoV-2 is crucial. Host cellular proteases, such as cathepsins and type II transmembrane serine proteases (TTSPs) have been reported to activate SARS-CoV-2 to promote their replication. Many members of the B-cell lymphoma-2 (Bcl-2) family proteins are involved in activating these host cellular proteases to promote IAV replication. 22,23,24,25 In the aftermath of the COVID-19 pandemic, many recent reviews have focussed on the similarities and differences between these respiratory viruses. 26,27,28 However, little is known about the role of Bcl-2 family proteinsactivated proteases in regulating SARS-CoV-2 replication. Consequently, this article compares the life cycle and mode of viral replication of IAV and SARS-CoV-2 and examines the potential roles of host Bcl-2 family proteins in SARS-CoV-2 replication to provide future research directions.

| Influenza A virus
IAV is a segmented negative-sense single-stranded RNA (ssRNA) virus belonging to the Orthomyxoviridae family. It consists of 8 gene segments enclosed in an enveloped virion which is 100 nm in diameter. 29,30 Haemagglutinin (HA), matrix (M), neuraminidase (NA), nucleoprotein (NP), non-structural protein (NS1), polymerase acidic protein (PA), polymerase basic protein 1 (PB1), and polymerase basic protein 2 (PB2) are the eight IAV gene segments which encode protein(s) with specific functions. 31,32 The segmented genome of IAV has important implications in virus evolution and immune escape. 33 The IAVs are divided into subtypes based on the sequences of HA and NA that are located on the surface of the virion envelope. 32 At least 18 HA types (H1 to H18) and 11 NA types (N1 to N11) have been identified so far. 34 IAVs infect various animal species such as birds, dogs, ferrets, horses, and pigs. Although, the main viral reservoirs, aquatic birds like waterfowls remain asymptomatic, IAVs pose a threat to humans as a zoonotic agent, generally causing mild infections. 35,36,37 However, some specific subtypes, like H1N1 and H5N1, cause severe illnesses/mortality in infected patients. 38 Further, crossover to other animals may lead to the emergence of pathogenic subtypes, such as the 1968 Hong Kong flu pandemic (H3N2) and the 2009 swine flu pandemic (H1N1) that caused severe disease. 39 IAVs that infect humans mainly belong to H1N1 and H3N2 sub-types. 40 IAVs causes both upper and lower respiratory tract infections and may attack other organs such as the central nervous system (CNS) and heart. 41 The mode of transmission of IAVs is mainly by respiratory droplets. Three routes of transmission (not mutually exclusive) have been postulated and are widely accepted: contact transmission (direct/indirect), large droplets, and respiratory droplets (or aerosols) albeit the relative contribution of each to the rapid spread and continued circulation of IAV is highly debatable. For IAV to cause infection in a host, a variety of factors related to the environment, the host, and the virus may play a role. 42 IAVs exhibit antigenic drift/shift properties, genetic phenomenon allowing them to avoid the host immune response via mutations. 27 Antigenic drift is the variation that occurs in antigen structures owing to point mutations in the HA and NA genes over time due to the lack of proofreading by viral RNA-dependent RNA polymerase (RdRp) usually leading to seasonal differences and epidemics. Antigenic shift, on the other hand, is the result of a sudden genetic reassortment between genome segments, which leads to the formation of a totally new virus and often leads to pandemics. 43 These antigenic drift/shift properties can potentially reduce the effectiveness of vaccines and become a considerable challenge in antiviral therapy. 44 Antiviral drugs like amantadine/rimantadine, 45 oseltamivir, and zanamivir 40 are used for prophylaxis and treatment of IAV infection; however, most human H1N1 and H3N2 viruses, including the 2009 H1N1 pandemic IAV, are resistant to these drugs and the frequency of resistance is increasing with time. 46,47,48,49 Consequently, currently vaccination is the most effective way to prevent IAV outbreaks.
Unfortunately, high incidences of mutation in both the HA and NA makes complete protection difficult. Presently, a universal flu vaccine that will be effective against any new IAV strain is the focus of flu researchers and hopefully, if achieved, will put an end to the yearly seasonal flu outbreaks. 50

| SARS-CoV-2
Coronaviruses (CoVs) are a group of viruses belonging to the Coronaviridae family and the Orthocoronavirinae subfamily. 51 63,64 The remaining one-third encodes 9 accessory proteins (ORFs) and 4 structural proteins; envelope (E), membrane (Memb), nucleocapsid (N), and spike (S). The SARS-CoV-2 genome is encapsidated by N, whereas M and E ensure its incorporation in the viral particle during the assembly process. S trimers protrude from the host-derived viral envelope and provide specificity for cellular entry receptors and have been shown to mediate viral entry into the host cells. 65 However, SARS-CoV-2-S is highly variable from SARS-CoV, sharing <75% nucleotide identity. 13 and TMPRSS4 may be involved in cleaving avian and human IAV HA proteins at an arginine residue which is essential for proteolytic activation of IAV HA subtypes. 71 Cleavage of the envelope glycoprotein HA by host proteases is a prerequisite for membrane fusion and essential for virus infectivity. 72 This suggests that TMPRSS2 may be a potential target for treatment of both influenza and coronavirus infections. Proteins in the viral ribonucleoprotein (vRNP) complex contain different nuclear localisation signals, thus helping the vRNP complex to enter the host cell nucleus via active transport. 73 The acidic environment of the endosome also activates M2 ion channel, hence acidifying the viral core, resulting in entrance of vRNP complex into the host cell. 74 Replication of viral genome does not require a primer but a full-length complementary RNA (cRNA), which is essential for the newly formed vRNP complex. The viral RNA polymerases first bind to the 3ʹ and 5ʹ-end of the segmented viral RNA and cRNA, respectively, then starts replication with the help of the 5ʹ cap of host pre-mRNAs via a PB1-PB2-mediated 'cap snatching' mechanism. 75 The conserved segment-specific nucleotides at the 3ʹ

| SARS-CoV-2
Epithelial cells in the nasal cavity are the primary site of attachment and replication of the inhaled SARS-CoV-2. The infectious agent then spreads and migrates across the respiratory tract, following the conducting airways. 86 The coronavirus invades two types of cells in the lungs, the mucus producing goblet cells and the ciliated cells. The cilia cells are the preferred viral host. 87 Coronavirus spike protein interacts with cellular receptors, angiotensin-converting enzyme 2 (ACE2), found in human cells in the heart, intestines, kidneys, and lungs. 88 The cell surface serine protease, TMPRSS2, promotes viral uptake and fusion at the cellular or endosomal membrane. SARS-CoV-2-S contains two receptor-binding domains (RBDs), S1 and S2, that mediates direct contact with ACE2, and an S1/S2 polybasic cleavage site that is proteolytically cleaved by cellular cathepsin L and TMPRSS2. 66,67,89 RBDs are used to bind with the ACE2 receptors, allowing the virion to penetrate eventually into the human cells by a process known as endocytosis. 90

| Influenza A virus
IAVs can facilitate the progress of its pathogenesis and transmission by inducing apoptosis in the airway epithelial cells, 24 natural killer cells, 92 and neutrophils. 93 Several viral proteins are involved in the regulation of host apoptosis. As viral replication must be completed before dismantling of the cell through apoptosis, the expression of the anti-apoptotic viral proteins may facilitate viral propagation prior to cell death. Activation of the apoptotic cascade has been shown to play a role in the processing of viral proteins and the maturation of viral particles. 94 The pro-apoptotic proteins promote IAV-induced apoptosis and virus replication 95,96,97 by facilitating the proper shuttling of viral genomic segments from the nucleus to the budding sites at the plasma membrane. In line with this, the multifunctional IAV protein NS1 has been implicated in suppressing the host interferon response, 98 and both promoting 99 and inhibiting 100 apoptosis.
Although the precise mechanism of how NS1 can limit apoptosis is not fully understood, it is quite possible that NS1 directly interacts with and inhibits pro-apoptotic host factors through a N-terminal F I G U R E 2 Schematic representation of the life/replication cycle of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). SARS-CoV-2 binds to its cognate receptor, angiotensin-converting enzyme 2 (ACE2), by specific interactions involving the viral envelope spike protein, which is followed by the virus-cell membrane fusion at the cell surface and endosomal compartments, and release of the RNA genome into the cytosol. The assembly of viral replication-transcription complexes leads to viral RNA synthesis which translates to viral structural proteins that are further inserted into the endoplasmic reticulum (ER). Genomic RNA is packaged into ribonucleoprotein complexes and virions are then assembled and transported out of the infected cells by exocytosis. SONI AND MEBRATU PDZ-binding motif. 100 It is interesting to note that the PDZ-binding motif of NS1 is also required for efficient viral propagation, as mutation of this domain can significantly reduce viral titres. 100 Hence, NS1 may prevent the early induction of apoptosis, consequentially aiding IAV pathogenesis.
Furthermore, IAV NP has been reported to induce host apoptosis to favour viral replication through interaction with ring finger 43 (RNF43) and apoptotic inhibitor 5 (API5) 101 or clusterin. 102 Even though the mechanism of NP-induced apoptosis is not fully elucidated yet, past reports have shown that NP can directly interact with the anti-apoptotic host factor API5, thereby preventing downregulation of APAF1 and promoting apoptosis. 103 NP exerts its proapoptotic function through the inhibition of the E3 ubiquitin ligase RNF43. 101 As RNF43 can mark p53 for degradation through ubiquitination, the interaction of NP with RNF43 can result in p53 stabilisation and consequently promote apoptosis through Bak/Bax activation. 101 Further, the IAV M1 has been reported to promote apoptosis by binding to heat shock protein 70, thus, activating the caspase cascade followed by apoptosis. 104 A recent transcriptomic study highlighted that apoptosis marker genes were expressed early in infection, at 8 h post infection (hpi), which is a host strategy to limit viral infection. The IAV PB1-F2 gene was suggested to be responsible for this induction. 105 PB1-F2 induced apoptosis and promoted viral replication through dysregulating mitochondrial potential. 106 Conclusively, these studies highlight the pro-viral role of apoptosis during the pathogenesis of IAV. 107 The role of these viral proteins in apoptosis suggests that they might be suitable targets for anti-IAV therapies. 63

| SARS-CoV-2
Recent novel studies on SARS-CoV-2 provide valuable insights into the possible role of cell death in the infection-induced tissue injury. 56 Apoptosis induction has been labelled as a hallmark of SARS-CoV-2 infection. 108 SARS-CoV-2 induces the apoptosis of the infected alveolar cells by using human lung stem cell-based alveolospheres. 109 Extensive apoptosis has been observed in lung epithelial cells of humanised ACE2 transgenic mice and Syrian hamsters. 34  proteins. 119 Emerging studies propose that SARS-CoV-2 infection-associated epithelial cell apoptosis plays a vital role in lungs disorder. 120

| BCL-2 FAMILY PROTEINS-ACTIVATED HOST CELLULAR PROTEASES AND THEIR ROLE IN IAV AND SARS-COV-2 REPLICATION
Several viruses, including IAVs, express proteins that undergo host cell caspase cleavage. 148 IAV-induced caspase activation causes a widening of nuclear pores to facilitate passive transport of vRNP to ensure efficient production of infectious virus progeny. 149,150 IAVs are structured into RNP segments consisting of viral RNA and viral proteins, the major one being the NP, a target of caspase cleavage. 24,151 NP acts as a shuttle for viral genomic segments from the nucleus to the budding sites at the plasma membrane, and localisation during the virus replication cycle affects virus titres. Following apoptotic stimuli, IAV NP or RNP has been shown to translocate partially to the cytoplasm in a caspase-3-dependent manner, 96 and nuclear retention of NP caused by a lack of caspase activity has been linked to decreased viral replication. 136 The pro-survival protein Bcl-2, on the other hand, inhibits IAV-induced apoptosis to mitigate viral replication rates. 135,152 Activation of caspase-3 during the cell death process is critical to the IAV life cycle 96  also impaired cytoplasmic export of vRNP, suggesting the potential for impaired shuttling of vRNP to the cytoplasm by Bik-deficiency. 24 It is not clear, however, whether the defects in the cleavage of viral proteins are caused by Bik-deficiency or increased expression of Bcl-2 contributes partially to the nuclear retention of viral NPs. Similarly, the viral ionic channel M2 protein is also cleaved by caspases in both human and avian influenza viruses 153,154 (Figure 3). Earlier studies also investigated the role of Bad, Bak, and Bax in IAV infection. 95,135 BAD promotes IAV replication through phosphorylation of BAD at S112 and S136 and apoptotic death of epithelial cells. 23 26 ). However, none of these antiviral approaches target Bcl-2 family proteins. Hence, IAV and/or SARS-CoV-2 proteinactivating host cell caspases may provide novel potential drug targets. 55 Inhibition of host factors (such as NP or M2-activating caspases) either by modulating the pro-apoptotic proteins (such as the Bik level) or by blocking the activity of specific caspases could be a novel approach to mitigate IAV replication ( Figure 3).

| PERSPECTIVES AND FUTURE DIRECTIONS
Both IAV and SARS-CoV-2 are enveloped RNA viruses and appear to have similar mode of infection, molecular mechanisms of replication, and transmission (reviewed in Ref. 27 ). The molecular mechanisms of IAV replication and pathogenesis have relatively been well explored.
Owing to the many similarities between these infectious agents, important lessons can be learnt from the knowledge available so far on IAV to lay a fundamental basis of understanding for SARS-CoV-2.
Antiviral drugs such as amantadine/rimantadine, 45 oseltamivir, and zanamivir, 40 are used for prophylaxis and treatment of IAV infection; however, most human H1N1 and H3N2 viruses, including the 2009 pandemic H1N1 IAV, are resistant to these drugs and the frequency of resistance is increasing steadily. 46,47,48,49 Although efforts are underway to find effective treatment for SARS-CoV-2, the recent appearances of new variants, such as delta 158  Inhibitors of host cell proteases have been used effectively to reduce viral replication and treat viral infections, such as in the case of HCV 157 and HIV-1. 155 The recent breakthroughs that led to the development of protease inhibitors, such as grazoprevir and elbasvir that are effective in treating hepatitis C 156,157 suggests that host cellular proteins can be good targets to treat infections caused by IAV and/or SARS-CoV-2. Host cellular proteases, such as caspases, cathepsins, and transmembrane proteases, such as TMPRSS2 have been implicated in the replication of both IAV and SARS-CoV-2 through cleaving and activating viral proteins to promote their replication. 24,148,160 For example, TMPRSS2 cleaves HA protein of IAVs and spike protein of SARS-CoV-2 to activate and promote their replication, suggesting that TMPRSS2 may be a potential target for the treatment of both influenza and coronavirus infections. In the case of IAVs, the pro-apoptotic members of the Bcl-2 family proteins mediate activation of caspases and cathepsins 24,148 that causes nuclear pore opening, 161 to allow efficient nucleocytoplasmic shuttling and proper assembly of components of viral proteins. 24  F I G U R E 4 Schematic representation highlighting putative anti-viral targets. Virus assembly and replication can be inhibited at several steps in the replication cycle of influenza A virus (IAV) and severe acute respiratory syndrome coronavirus-2. Blocking pro-apoptotic Bcl-2 family proteins like Bcl-2 interacting killer (Bik) or inhibiting proteases like caspases, cathepsins, and transmembrane proteases may impair cleavage of viral proteins mitigating viral replication and pathogenicity. Further, inhibitors may impair the nucleocytoplasmic transport of viral ribonucleoprotein (vRNP) to prevent proper assembly of progeny virions resulting in reduced viral replication.